The present invention relates to a thermostable creatine amidinohydrolase and a process for producing the same.
Creatine amidinohydrolase is an enzyme catalyzing hydrolysis of creatine to give sarcosine and urea, which can be used to determine an amount of creatine in human serum or urine, being usable as a diagnostic agent for various diseases such as kidney diseases and the like.
For the reason that a creatine amidinohydrolase from Alcaligenes having Km value of approximately 13 mM and thermostability of around 45xc2x0 C. shows a lower Km value, and moreover, taking into consideration the distribution and the sales of the agent in liquid form, a creatine amidinohydrolase having stability at an increased temperature has been sought. Furthermore, conventionally, attempts to improve the thermostability of creatine amidinohydrolase by genetic modification frequently ended with the results showing the same Km value as an original strain or even a decreased value, and there have been no cases where the thermostability and the Km value thereof were improved.
The object of the present invention is to provide a thermostable creatine amidinohydrolase and a process for producing the same.
The present inventors have further studied to solve the above problem, and found that a thermostable creatine amidinohydrolase was obtainable by modifying a gene of an Alcaligenes creatine amidinolhydrolase (JP-A-8-89255 (1996)), thus completing the present invention.
In one aspect, the present invention provides an isolated thermostable creatine amidinohydrolase having the following physicochemical properties:
(a) hydrolyzing 1 mol of creatine to give 1 mol of sarcosine and 1 mol of urea;
(b) having a substrate specificity to creatine;
(c) having an optimum pH range of 7.0 to 8.0;
(d) having a stable pH range of 4.0 to 11.0;
(e) having the optimum temperature of around 45xc2x0 C.;
(f) being thermostable at 53xc2x0 C.; and
(g) having a molecular weight of 92,000 Da (as determined by gel filtration).
In an embodiment, the thermostable creatine amidinohydrolase of the present invention is derived from an Alcaligenes bacterium.
In another embodiment, the thermostable creatine amidinohydrolase of the invention is generated by introducing a mutation(s) into a gene of the Alcaligenes creatine amidinohydrolase and allowing expression of the obtained genetic mutant.
In another embodiment, the thermostable creatine amidinohydrolase of the invention has an amino acid sequence comprising a mutation(s) relative to the amino acid sequence of SEQ ID NO:1. The term xe2x80x9cmutationxe2x80x9d as used herein means at least one amino acid substitution, addition, insertion, or deletion.
Furthermore, in another embodiment, the thermostable creatin amidinohydrolase of the invention can be produced by an E.coli JM109 strain (pUCE100 B-40) (Accession Number: FERM BP-6867) or a strain derived therefrom.
In another aspect, the invention provides a process for producing the thermostable creatine amidinohydrolase which comprises incubating a microorganism capable of producing the above amidinohydrolase, and recovering said amidinohydrolase from the culture.
In another embodiment of the invention, said microorganism is an E.coli JM109 strain (pUCE100 B-40) (Accession Number: FERM BP-6867) or a strain derived therfrom.
The term xe2x80x9cstrain derived therefromxe2x80x9d as used herein means a mutant which can be obtained by natural or artificial mutagenesis of E.coli JM109 (pUCE100 B-40), the mutant being a strain capable of producing the thermostable creatine amidinohydrolase with physicochemical properties equivalent to those mentioned above.